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P-body and Stress Granule Quantification in Caenorhabditis elegans
秀丽隐杆线虫中P-body和应激颗粒的定量   

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Abstract

Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules is Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA degradation, translational repression and cellular mRNA transport (Sheth and Parker, 2003). Another type of RNP granules, the stress granules (SGs), majorly contain mRNAs associated with translation initiation factors and are formed upon stress-induced translational stalling (Kedersha et al., 2000 and 1999). Multiple evidence obtained from studies in unicellular organisms supports a model in which P bodies and SGs physically interact during cellular stress to direct mRNAs for transport, decay, temporal storage or reentry into translation (Anderson and Kedersha, 2008; Decker and Parker, 2012). The quantification, distribution and colocalization of P bodies and/or SGs are essential tools to study the composition of RNP granules and their contribution to fundamental cellular processes, such as stress response and translational regulation. In this protocol we describe a method to quantify P bodies and SGs in somatic tissues of the nematode Caenorhabditis elegans.

Keywords: Caenorhabditis elegans(秀丽隐杆线虫), mRNP granules(mRNP颗粒), Processing bodies(P 小体), Stress granules(应激颗粒), Transgenesis(转基因)

Background

Thus far, most protocols to study P bodies and SGs were developed for yeast or human cell lines (Buchan et al., 2010). Little is known about the function of somatic RNP granules in multicellular organisms. The simple model organism C. elegans has been extensively used to study germline-specific P granules, which are distinct from P bodies and SGs, and important structures for germline development and function (Updike and Strome, 2010). Although the principles of the presented procedure can be applied to count germline-specific P granules, the protocol focusses on the quantification of somatic RNP granules. Several studies have identified a conserved function of somatic P bodies in the translational deregulation via miRNA pathways in C. elegans (Ding et al., 2005; Zhang et al., 2007). More recently, various tools were created to study the involvement of cytoplasmic RNP granules in cellular and organismal stress response, development and ageing in the nematode (Cornes et al., 2015; Huelgas-Morales et al., 2016; Rieckher et al., 2015; Rousakis et al., 2014; Sun et al., 2011; Table 1).

Such studies take advantage of the comparatively easy implementation of transgenesis methods in C. elegans that allow to constitutively express fluorescent fusion proteins (e.g., green fluorescent protein [GFP]), endogenously or in specific tissues (Rieckher et al., 2009). A collection of fosmids carrying gfp-tagged P body- and SG-specific genes can be obtained at the ‘C. elegans TransGeneome’ project, a genome-scale transgenic project for fluorescent- and affinity-tagged proteins for expression in the nematode (Sarov et al., 2012; Table 1). C. elegans is transparent, which allows for efficient application of fluorescence microscopy methods that are easily combined with differential interference contrast (DIC) microscopy to reveal fluorescent protein expression in an anatomical context. Mounting transgenic animals for P body and SG imaging is based on a previously described method using nanoparticles for immobilization (Kim et al., 2013), since commonly applied anesthetics in C. elegans can induce stress, resulting in increased RNP granule formation. Fluorescence-tagged P body or SG-intensity can be imaged by epifluorescence light microscopy (see Procedure C), while fluorescence intensity, a detailed count and size measurements of P bodies and SGs can be obtained via confocal laser scanning microscopy (see Procedure D).

Table 1. Tools available for transgenic expression of P body/SG-factors in C. elegans

*available at CGC
+germline-specific promoter fusion
$C. elegans TransGeneome project (Sarov et al., 2012)
&can be found in both types of RNP granules

Materials and Reagents

  1. Sterile pipette tips
  2. Surgical disposable scalpel (Braun Medical, catalog number: 5518075 )
  3. Worm Pick with platinum wire (Genesee Scientific, catalog numbers: 59-30P6 )
  4. Pre-flattened tip (Genesee Scientific, catalog numbers: 59-AWP )
  5. Microscope slides (76 x 26 mm) (Carl Roth, catalog number: 0656.1 )
  6. Cover slips (24 x 24 mm) (Carl Roth, catalog number: H875.2 )
  7. Tape (~1 mm thickness)
  8. Greiner Petri dishes (60 x 15 mm) (Greiner Bio One, catalog number: 628161 )
  9. C. elegans strains (see Table 1 for available transgenes)
  10. Escherichia coli OP50 strain (obtained from the Caenorhabditis Genetics Center)
  11. Nail polish
  12. Polystyrene beads (Polybead, 2.5% by volume, 0.1 µm diameter)
  13. Potassium dihydrogen phosphate (KH2PO4) (Carl Roth, catalog number: P018.1 )
  14. Di-potassium hydrogen phosphate (K2HPO4) (Carl Roth, catalog number: 5066.1 )
  15. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  16. Di-sodium hydrogen phosphate (Na2HPO4) (Carl Roth, catalog number: T876.1 )
  17. Bacto peptone (BD, catalog number: 211677 )
  18. Streptomycin sulfate salt (Sigma-Aldrich, catalog number: S6501 )
  19. Agar (Sigma-Aldrich, catalog number: 05040 )
  20. Cholesterol stock solution (SERVA Electrophoresis, catalog number: 17101.01 )
  21. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  22. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506 )
  23. Nystatin stock solution (Sigma-Aldrich, catalog number: N3503 )
  24. Agarose (Biozym, catalog number: 840004 )
  25. Phosphate buffer (1 M; sterile) (see Recipes)
  26. Nematode growth medium (NGM) agar plates (see Recipes)
  27. M9 buffer (see Recipes)
  28. 5% agarose pads (see Recipes)

Equipment

  1. Dissecting stereomicroscope (Olympus, model: SMZ645 )
  2. Epifluorescence microscope (ZEISS, model: Axio Imager Z2 , objective EC Plan-Neofluar 10x/0.3)
  3. Confocal microscope (we use the Zeiss LSM710 confocal microscope with an Argon multiline laser source 25 mW and a tunable laser with the wavelength range 488-640 nm) (ZEISS, model: LSM710)
  4. Microwave
  5. Incubators for stable temperature (AQUA®LYTIC incubator 20 °C)
  6. Scale
  7. Cylindrical glass beaker (25 ml) (VWR, catalog number: 213-1120 )
  8. Autoclave

Software

  1. ZEN 2009 software (or later), Carl Zeiss AG, Jena, Germany (or any other software controlling a fluorescence microscope or confocal microscope)
  2. Microsoft Office 2011 Excel (Microsoft Corporation, Redmond, USA)
  3. Fiji or ImageJ (https://fiji.sc/ or https://imagej.nih.gov/ij/)

Procedure

  1. Growth and synchronization of transgenic C. elegans population
    1. When working with a transgenic strain with integrated genetic array (Table 1), use a sterile pipette tip (200 µl) to cut a small chunk (0.5 x 0.5 cm) of agar containing animals from an older plate and transfer it to a freshly Escherichia coli (OP50) seeded NGM plate (see Recipes). When working with a transgenic line with a non-integrated extrachromosomal array, pick ~25 L4 larvae or adult transgenic animals based on the selection-marker to a freshly OP50 seeded NGM plate.
    2. Incubate the nematodes at the standard temperature of 20 °C.
    3. 3.5 days later the plates contain a mixed population of larval stages.
    4. Synchronize nematodes by picking ~30 transgenic animals in L4 stage under a dissecting stereomicroscope and transfer them onto separate OP50 seeded plates. The L4 stage can be identified based on the presence of a half-moon shaped light structure in the area of the vulva and the relative size of the animal (Figure 1).
    5. Grow the animals for 24 h at 20 °C into day 1 adults and proceed with mounting the sample (Procedure B).


      Figure 1. Identification of C. elegans larval stages in a mixed population. The image depicts all 4 larval stages, young adult and day 1 adult as seen under a dissecting stereomicroscope. The arrow indicates the half-moon shaped structure in the area of the vulva, which is indicative for the L4 larval stage.

  2. Mounting animals for imaging
    1. Prepare fresh 5% agarose pads (see Recipes).
    2. Pipette 3 µl polystyrene beads suspension in the center of the agarose pad.
    3. Use a platinum wire to pick ~30 transgenics from the OP50 seeded plates into the polystyrene bead suspension.
    4. Gently place a coverslip on top of the agarose/worm suspension and proceed with the imaging procedure. Optionally, the agarose pads can be sealed with nail polish, which will retain humidity for long-term (> 3 h) imaging.
    5. Animals can be recovered from unsealed plates by gently lifting one edge of the coverslip with the help of a scalpel and adding 10 µl of M9 buffer to suspend the worms. Subsequently, worms can be transferred with an eyelash to OP50-seeded NGM plates.

  3. Imaging P body/SG number with an epifluorescence light microscope
    1. Start up the light microscope and imaging software (ZEN, Zeiss).
    2. Place the agarose pad on the imaging stage of the fluorescent light microscope and locate the animals. Use a 10x objective to image the whole animal.
    3. Use the DIC channel to focus on an anatomical landmark. Most appropriate are the grinder and the lumen of the pharynx, which are located in the most central transverse (horizontal) position within the animal (Figure 2A).
    4. Change to the fluorescent channel to visualize P body or SG-specific fluorescence and define exposure time and fluorescent light intensity. These parameters depend on the transgene and have to be defined empirically. Saturation of the fluorescent signal has to be avoided by lowering exposure time and/or fluorescent light intensity. However, too short exposure might result in lowered sensitivity of detecting RNP granules (Figure 2B). For transgenes that co-express various reporters for P bodies/SGs repeat this step in the corresponding fluorescent channel.
    5. Once the best imaging conditions are determined, take a snapshot of all fluorescent channels and the DIC channel. Take images of at least 25 animals to obtain enough data for statistical analysis.
    6. Save the data as an image stack. Zeiss microscopes produce *.czi file formats, which can be processed by ImageJ/Fiji freeware (see Data analysis). However, images can also be stored/exported and further processed in any other common format including *.jpg, *.png, *.tiff, etc., and then proceed with the data analysis.


      Figure 2. Representative images of epifluorescence images of an adult transgenic animal expressing the P body reporter DCAP-1::dsRED. Images were recorded with an Axio Imager Z2 through a 10x magnification objective. Size bars are 100 µm. A. DIC image. The inlay shows a digitally enhanced view on the pharynx. The arrow points out the grinder, which has to be in focus before switching to the fluorescent channel. B. Fluorescent image of the P body reporter DCAP-1::dsRED. C. Screenshot of image processing. Choose the polygon selection tool to surround the whole animal. Open Analyze > Measure to get area size and MPI. (Full genotype of transgenic animal: N2;Ex[pdcap-1DCAP-1::dsRED; pRF4] published in Rieckher et al., 2015.)

  4. Imaging P body/SG number, dynamics and colocalization with a confocal microscope
    1. Start up the confocal microscope and corresponding imaging software.
    2. Activate the appropriate laser(s) for fluorescent detection of P body/SG reporters.
    3. Locate the animal through the eyepiece with a 10x objective using transmission light and center the field of view on the area of interest (e.g., the pharynx).
    4. Upon localization, switch to the 40x objective and readjust the focus.
    5. In the confocal operating software (acquisition) design a protocol for a z-stack scan.
      1. Different RNP granules significantly vary in size and intensity across different transgenes and within samples (Teixeira et al., 2005). Hence, a balance has to be established empirically between laser power and gain (voltage of photomultiplier tubes [PMTs]/detectors) to set the highest and lowest detection limit. If available, use the Smart Setup as a starting point. Try to avoid overexposure but test the lower detection limit by stepwise increasing laser power and/or gain. The final settings have to be reproducibly applied to all samples. For the example in Figure 2 we use the tunable laser at 570 nm (excitation dsRED), laser power 3%, and Master gain 580.
      2. Keep the pinhole size for an optimal section thickness at 1 AU.
      3. The distance between z-stacks (slices) should be maximum 1.5 µm. Decreasing z-stack distance might enhance the capability to detect smaller RNP granules but increases photobleaching. Cover a similar volume in each scanned sample. We perform 30 z-stacks at 1.5 µm across the pharyngeal region (Figures 2 and 3).
      4. Assign the frame size (resolution). We use 1024 x 1024 (Figures 2 and 3).
      5. Use the transmitted light detector (T-PMT) to produce a DIC/Brightfield image in parallel (optional).
    6. Once the best imaging conditions are determined, perform a scan of the fluorescent channel. When imaging different P bodies/SGs co-expressed in the same transgene use sequential scan for additional fluorescent channels. Take datasets of at least 15 animals to obtain enough data for statistical analysis.
    7. Upon completion of the scan use the processing mode of the ZEN software to produce a maximum intensity projection (MIP), summarizing the fluorescence intensity of all stacks in one image (Figures 3A and 3B). Consistently save as *.czi file or other image file format. Proceed with data analysis. Alternatively, single stacks or subsets of stacks can be processed and analyzed.


      Figure 3 Representative images of the pharyngeal region of an adult transgenic animal expressing the P body reporter DCAP-1::dsRED and the SG reporter IFE-2::GFP. Images were recorded with an LSM710 confocal microscope and processed into MIP. Size bars are 50 µm. A. P body reporter DCAP-1::dsRED expression. B. SG reporter IFE-2::GFP. C. Screenshot of image processing via Fiji/ImageJ. Use the polygonal tool to surround the area of interest, assign a threshold and use Analyze Particles to obtain information about P body/SG intensity, number and size. (Full genotype of transgenic animal:N2;Ex[pdcap-1DCAP-1::dsRED; pife-2IFE-2::GFP; pRF4] published in Rieckher et al., 2015.)

Data analysis

  1. Processing P body- and SG-data with Fiji (ImageJ)
    1. Download and install the Fiji freeware from https://fiji.sc/.
    2. Open an image file from fluorescence microscopy or a MIP file from confocal laser scanning microscopy by dragging/dropping into the Fiji software.
    3. Images will open as stacks of DIC and the fluorescent channels that were recorded. In the control panel of Fiji chose Image > Stacks > Stack to images to split the channels.
    4. Change Image > Type > 8 bit for further analysis steps.
    5. Chose the image of the fluorescent channel that should be analyzed and apply the polygon selection tool to further narrow down the region of interest (ROI). For images received from fluorescence microscopy encircle the whole animal (Figure 2C). In MIP images a specific region, such as the pharynx, should be selected (Figure 3C).
    6. For epifluorescence images choose ‘Analyze > Measure’ to obtain a summary about the Area size and the mean pixel intensity (MPI) in arbitrary units (AU) of the assigned area. This information will be displayed in a separate window and can be copied/pasted directly from there in a data analysis software and/or saved as an excel file (*.xls).
    7. For confocal images proceed with the measurement of P body and SG number and size a threshold has to be determined that assigns single pixels or pixel clusters to define fluorescent RNP granules. Chose Image > Adjust > Threshold and empirically assign a proper Min/Max value (Figure 3C). This value will vary depending on the transgene, choice of microscope and the imaging settings. Once assigned, these values have to stay consistent throughout every analysis.
    8. Chose Analyze > Analyze Particles and tick ‘Display results’, ‘Summarize’ and ‘Exclude on Edges’ and press OK (Figure 3C).
    9. Two separate Windows display the ‘Results’ for every RNP granule, producing Area (Granule size) and Mean (MPI), and the ‘Summary’ including ‘Count’ (RNP granule number), ‘Total Area’ and ‘Average Size’. These data can be copied directly into a data analysis software and/or saved as excel file.
  2. Statistical analysis
    1. Stay consistent with the number of animals examined for each strain and condition.
    2. Each assay should be repeated at least three (3) times.
    3. Use the Mann Whitney or Wilcoxon Kruskal Wallis test with a significance cut-off level of P < 0.05 for comparisons between different groups and correct for multiple pairwise comparisons using Bonferroni or False Discovery Rate (FDR).

Notes

P bodies and SGs are known to substantially vary in size and number across cells (Teixeira et al., 2005). Measurements become more precise and final values underlie less standard deviation when focusing on quantification in specific tissues (e.g., muscles, pharynx, or intestine) and increasing sample size.

Recipes

  1. Phosphate buffer (1 M)
    1. For 1 L, dissolve 102.2 g KH2PO4 and 57.06 g K2HPO4 in distilled water and fill up to 1 L. This is a 1 M solution, pH 6.0
    2. Autoclave at 121 °C for 20 min
    3. Store at room temperature
  2. Nematode growth medium (NGM) agar plates
    1. Mix 3 g NaCl, 2.5 g Bacto peptone, 0.2 g streptomycin, 17 g agar and add 900 ml distilled water. Autoclave at 121 °C for 20 min
    2. Let cool to 55-60 °C
    3. Add 1 ml cholesterol stock solution, 1 ml 1 M CaCl2, 1 ml 1 M MgSO4, 1 ml nystatin stock solution, 25 ml sterile 1 M phosphate buffer, pH 6.0, and distilled sterile water up to 1 L
    4. Pipette 8 ml medium per Petri dish and leave to solidify
    5. Store the plates at 4 °C until use
  3. M9 buffer
    1. Dissolve 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl in 1 L distilled water
    2. Autoclave at 121 °C for 20 min
    3. Let cool and add 1 ml 1 M MgSO4 (sterile)
    4. Store M9 buffer at 4 °C
  4. 5% agarose pads
    1. Weigh 0.5 g agarose and add it into a cylindrical glass beaker
    2. Add 10 ml M9 buffer
    3. Heat the mixture in a microwave until it is close to boiling. Take it out, stir with a pipette tip and boil again. Repeat until the agarose is dissolved completely
    4. Modify two microscope slides by putting a stripe of tape along the midline
    5. Place an empty microscope slide between two taped slides (Figure 4A)
    6. Put a drop (ca. 50 µl) of fresh 5% agarose solution in the middle of the slide (Figure 4B)
    7. Take a fourth microscope slide and place it on top of the agarose drop. Gently press down to flatten the drop. The tape serves as spacer to give the agarose pad a specific thickness (Figure 4C)
    8. Let the agarose harden for 30 sec and remove the top microscope slide
    9. Quickly cut the edges of the agarose pad with the scalpel to a square of approximately 20 x 20 mm
    10. Immediately proceed with the sample preparation (see Procedure B), since the agarose pads will start drying within approximately 5 min
      Note: Leaving the top microscope slide as a cover contains the humidity longer (approx. 1 h). Thus, several agarose pads can be prepared and used swiftly during the experiments.


      Figure 4. Preparation of agarose pads for fluorescent imaging in C. elegans. A. Two glass slides modified with tape flank an empty microscope slide. B. Place a drop of 50 µl 5% agarose solution in the middle of the microscope slide (arrow). C. Swiftly put another microscope slide on top and gently push it down to flatten the agarose drop.

Acknowledgments

This work was funded by grants from the European Research Council (ERC), the European Commission 7th Framework Programme. We want to acknowledge the Fang-Yen laboratory, Philadelphia, PA, who developed the agarose pads for long-term imaging of C. elegans.

References

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简介

真核细胞含有各种类型的细胞质,非膜结合的核糖核蛋白(RNP)颗粒,其由非翻译mRNA和多种相关蛋白组成。一种突出类型的RNP颗粒是加工体(P体),其主要包含翻译失活的mRNA和介导mRNA降解,翻译抑制和细胞mRNA运输的蛋白质阵列(Sheth和Parker,2003)。另一种类型的RNP颗粒,应激颗粒(SGs​​)主要含有与翻译起始因子相关的mRNA并且在应激诱导的翻译失速(Kedersha等人,2000和1999)中形成。从单细胞生物学研究中获得的多个证据支持了一种模型,其中P体和SG在细胞应激期间物理相互作用以将mRNA转运,转移,临时储存或折返到翻译中(Anderson和Kedersha,2008; Decker和Parker,2012)。 P体和/或SG的量化,分布和共定位是研究RNP颗粒的组成及其对基础细胞过程(如压力反应和翻译调节)的贡献的重要工具。在这个协议中,我们描述了一种量化线虫秀丽隐杆线虫体细胞中P体和SGs的方法。

背景 到目前为止,研究P体和SG的大多数方案是针对酵母或人类细胞系开发的(Buchan et al。,2010)。对多细胞生物体内的体细胞RNP颗粒的功能知之甚少。简单的模型生物体。线虫已广泛用于研究与P体和SG不同的种系特异性P颗粒,以及种系发育和功能的重要结构(Updike和Strome,2010)。虽然所提出的方法的原理可以应用于计数种系特异性P颗粒,但该方案集中于体细胞RNP颗粒的定量。几项研究已经确定了在C中通过miRNA途径的翻译失调的体细胞P体的保守功能。 elegans (Ding等人,2005; Zhang等人,2007)。最近,创建了各种工具来研究胞质RNP颗粒在细胞和生物应激反应,线虫发育和衰老中的参与(Cornes等人,2015; Huelgas-Morales et al。 2011年; Rieckher等人,2015年; Rousakis等人,2014年; Sun等人,2011年,2011年; ; 表格1)。
&nbsp;这种研究利用在C中比较容易地实现转基因方法。允许组成型表达荧光融合蛋白(例如,绿色荧光蛋白[GFP]),内源性或特定组织中的线虫(Rieckher等人,2009) )。携带 gfp 标签的身体和SG特异性基因的载体的集合可以在“C”获得。 elegans TransGeneome'项目,一种用于荧光标记和亲和标记的蛋白质在线虫中表达的基因组规模转基因项目(Sarov等人,2012;表1)。 C。线虫是透明的,其允许有效应用荧光显微镜方法,其容易与差异干涉对比(DIC)显微镜组合以在解剖学上下文中显示荧光蛋白表达。安装用于P体和SG成像的转基因动物基于使用纳米颗粒进行固定的先前描述的方法(Kim等人,2013),因为常规应用的麻醉剂在C中。线虫可诱导应激,导致RNP颗粒形成增加。荧光标记的P体或SG-强度可以通过落射荧光光学显微镜(参见方法C)成像,而荧光强度,P体和SG的详细计数和尺寸测量可以通过共聚焦激光扫描显微镜获得(参见方法D) 。

表1.在C中转基因表达P体/ SG-因子的工具。 elegans

CGC可以使用 *
+ 种系特异性启动子融合
$ C。 elegans TransGeneome项目(Sarov 等人,2012)
可以在两种类型的RNP颗粒中找到

关键字:秀丽隐杆线虫, mRNP颗粒, P 小体, 应激颗粒, 转基因

材料和试剂

  1. 无菌移液器吸头
  2. 手术一次性手术刀(Braun Medical,目录号:5518075)
  3. 蠕虫选择铂丝(Genesee Scientific,目录号:59-30P6)
  4. 预先扁平的提示(Genesee Scientific,目录号:59-AWP)
  5. 显微镜载玻片(76 x 26 mm)(Carl Roth,目录号:0656.1)
  6. 盖板(24 x 24 mm)(Carl Roth,目录号:H875.2)
  7. 胶带(〜1 mm厚)
  8. Greiner Petri菜(60 x 15毫米)(Greiner Bio One,目录号:628161)
  9. C。线虫菌株(见表1可得转基因)
  10. 大肠杆菌OP50菌株(从Caenorhabditis遗传学中心获得)
  11. 指甲油
  12. 聚酯珠(Polybead,2.5体积%,0.1μm直径)
  13. 磷酸二氢钾(KH 2 PO 4)(Carl Roth,目录号:P018.1)
  14. 磷酸氢二钾(K 2 HPO 4)(Carl Roth,目录号:5066.1)
  15. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  16. 磷酸氢二钠(Na 2 HPO 4)(Carl Roth,目录号:T876.1)
  17. Bacto蛋白胨(BD,目录号:211677)
  18. 硫酸链霉素盐(Sigma-Aldrich,目录号:S6501)
  19. 琼脂(Sigma-Aldrich,目录号:05040)
  20. 胆固醇储备溶液(SERVA Electrophoresis,目录号:17101.01)
  21. 氯化钙脱水(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  22. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M7506)
  23. 制霉菌素储备溶液(Sigma-Aldrich,目录号:N3503)
  24. 琼脂糖(Biozym,目录号:840004)
  25. 磷酸盐缓冲液(1M;无菌)(参见食谱)
  26. 线虫生长培养基(NGM)琼脂平板(参见食谱)
  27. M9缓冲区(见配方)
  28. 5%琼脂糖垫(见食谱)

设备

  1. 解剖立体显微镜(Olympus,型号:SMZ645)
  2. 荧光显微镜(ZEISS,型号:Axio Imager Z2,客观EC Plan-Neofluar 10x/0.3)
  3. 共聚焦显微镜(我们使用Zeom LSM710共焦显微镜,氩弧多激光源25 mW,波长范围488-640 nm的可调谐激光)(ZEISS,型号:LSM710)
  4. 微波
  5. 温度稳定的孵化器(AQUA ® LYTIC培养箱20°C)
  6. 比例
  7. 圆柱形玻璃烧杯(25ml)(VWR,目录号:213-1120)
  8. 高压灭菌器

软件

  1. ZEN 2009软件(或更高版本),德国耶拿Carl Zeiss AG(或控制荧光显微镜或共焦显微镜的任何其他软件)
  2. Microsoft Office 2011 Excel(Microsoft Corporation,Redmond,USA)
  3. 斐济或ImageJ( https://fiji.sc/ https://imagej.nih.gov/ij/

程序

  1. 转基因C的生长和同步。线虫人口
    1. 当使用具有整合遗传数组的转基因菌株(表1)时,使用无菌移液器吸头(200μl)从较旧的板切割含有小块(0.5×0.5cm)的含琼脂的动物,并将其转移到新鲜的大肠杆菌(OP50)种子NGM板(参见食谱)。当使用具有非整合的染色体外阵列的转基因品系时,根据选择标记将约25LL幼虫或成年转基因动物挑选到新鲜OP50种子NGM板。
    2. 在20℃的标准温度下孵育线虫。
    3. 3.5天后,板块含有混合的幼虫阶段。
    4. 通过在解剖立体显微镜下在L4阶段挑选〜30个转基因动物并将其转移到单独的OP50种子板上来同步线虫。可以根据外阴区域中的半月形光结构和动物的相对大小(图1)来确定L4阶段。
    5. 在20°C将动物生长24小时到第1天成年人,并继续安装样品(程序B)

      图1.识别C。线虫混合人群中的幼虫阶段。该图像描绘了在解剖立体显微镜下所见的所有4个幼虫阶段,年轻成年人和第1天的成年人。箭头表示外阴区域的半月形结构,表示L4幼虫阶段。

  2. 安装动物成像
    1. 准备新鲜的5%琼脂糖垫(见食谱)。
    2. 在琼脂糖垫的中心吸移3μl聚苯乙烯珠悬浮液。
    3. 使用铂丝从OP50接种板中挑取〜30个转基因进入聚苯乙烯珠悬浮液。
    4. 轻轻地将盖玻片放在琼脂/蠕虫悬浮液的顶部,然后继续进行成像程序。任选地,琼脂糖垫可以用指甲油密封,其将保持长时间(> 3小时)成像的湿度。
    5. 动物可以从未密封的平板上通过在手术刀的帮助下轻轻抬起盖玻片的一个边缘并加入10μlM9缓冲液来悬挂蠕虫。随后,蠕虫可以用睫毛转移到OP50种子的NGM板上。

  3. 用成像光学显微镜成像P体/SG数
    1. 启动光学显微镜和成像软件(ZEN,Zeiss)。
    2. 将琼脂糖垫放置在荧光显微镜的成像阶段,并定位动物。使用10x的目标来形象整个动物。
    3. 使用DIC通道将焦点集中在一个解剖学的地标上。最合适的是位于动物中最中心的横向(水平)位置处的咽的研磨器和内腔(图2A)。
    4. 切换到荧光通道,以观察P体或SG特异性荧光,并定义曝光时间和荧光强度。这些参数取决于转基因,必须用经验定义。必须通过降低曝光时间和/或荧光强度来避免荧光信号的饱和度。然而,太短的暴露可能导致检测RNP颗粒的灵敏度降低(图2B)。对于共同表达P体/SG的各种记者的转基因在相应的荧光通道中重复该步骤。
    5. 一旦确定了最佳成像条件,就会获取所有荧光通道和DIC通道的快照。拍摄至少25只动物的图像,以获得足够的数据进行统计分析。
    6. 将数据保存为图像堆栈。蔡司显微镜产生* .czi文件格式,可由ImageJ/Fiji免费软件处理(参见数据分析)。然而,图像也可以以任何其他通用格式(包括* .jpg,* .png,* .tiff,等)存储/导出和进一步处理,然后继续进行数据分析。 />

      图2.表达P体记者DCAP-1 :: dsRED的成年转基因动物的表面荧光图像的代表性图像。用Axio Imager Z2通过10x放大目标记录图像。尺寸棒为100μm。 A. DIC图像。嵌体显示了咽部上的数字增强视图。箭头指出研磨机,在切换到荧光通道之前必须对焦。 B. P体记者DCAP-1 :: dsRED的荧光图像。 C.图像处理的截图。选择多边形选择工具以围绕整个动物。打开分析>测量以获得面积大小和MPI。 (转基因动物的全基因型:N 2;出版于Rieckher等人,2015年的Ex [p dcap-1 DCAP-1 :: dsRED; pRF4])

  4. 成像P体/SG数,动力学和colocalization与共聚焦显微镜
    1. 启动共焦显微镜和相应的成像软件。
    2. 激活适用于P体/SG记者荧光检测的激光。
    3. 通过目镜使用传输光将目标物体定位在10x物镜上,并将感兴趣区域(例如,咽部)置于视野的中心。
    4. 本地化后,切换到40x目标并重新调整焦点。
    5. 在共焦操作软件(采集)中设计了一个z-stack扫描协议。
      1. 不同的RNP颗粒在不同转基因和样品中的大小和强度都有显着变化(Teixeira等,2005)。因此,必须在激光功率和增益(光电倍增管[PMTs /检测器的电压]之间根据经验建立平衡,以设置最高和最低检测限。如果可用,请使用智能设置作为起点。尽量避免过度暴露,但通过逐步增加激光功率和/或增益来测试较低的检测限。最终设置必须可重复地应用于所有样品。对于图2中的示例,我们使用570 nm(激发dsRED),激光功率3%和主增益580的可调激光器。
      2. 将针孔尺寸保持在1 AU的最佳截面厚度。
      3. z-叠层(切片)之间的距离应该最大为1.5μm。减小z-叠层距离可增强检测较小的RNP颗粒的能力,但增加光漂白。在每个扫描样品中覆盖相似的体积。我们在咽部区域(图2和图3)中以1.5μm的速度进行30个z-叠层。
      4. 分配帧大小(分辨率)。我们使用1024 x 1024(图2和3)。
      5. 使用透射光检测器(T-PMT)并行产生DIC/Brightfield图像(可选)。
    6. 一旦确定了最佳成像条件,就执行荧光通道的扫描。当在相同的转基因中共表达的不同P体/SG成像时,使用顺序扫描来获得额外的荧光通道。获取至少15只动物的数据,以获得足够的数据进行统计分析。
    7. 扫描完成后,使用ZEN软件的处理模式产生最大强度投影(MIP),总结了一个图像中所有叠层的荧光强度(图3A和3B)。一致保存为* .czi文件或其他图像文件格式。继续进行数据分析。或者,可以处理和分析单个堆栈或堆栈子集。


      图3表示P体报告者DCAP-1 :: dsRED和SG报告子IFE-2 :: GFP的成年转基因动物的咽部区域的代表性图像。使用LSM710共焦显微镜记录图像并处理成MIP。尺寸棒为50μm。 A.P体记者DCAP-1 :: dsRED表达。 B. SG记者IFE-2 :: GFP。 C.通过斐济/ImageJ进行图像处理的截图。使用多边形工具围绕感兴趣区域,分配阈值,并使用"分析粒子"获取有关P体/SG强度,数量和大小的信息。 (转基因动物的全基因型:N 2;出版于Rieckher等人的Ex [p dcap-1 DCAP-1 :: dsRED; pife-2IFE-2 :: GFP; pRF4]/em>,2015)

数据分析

  1. 用斐济(ImageJ)处理P身体和SG数据
    1. https://fiji.sc/下载并安装斐济免费软件。
    2. 从共焦激光扫描显微镜通过拖放到斐济软件中,从荧光显微镜或MIP文件打开图像文件。
    3. 图像将作为DIC和记录的荧光通道的堆叠打开。在斐济的控制面板中选择了Image>堆叠>堆叠到图像以分割通道。
    4. 更改图像>类型> 8位进一步分析步骤。
    5. 选择应分析的荧光通道的图像,并应用多边形选择工具进一步缩小感兴趣区域(ROI)。对于从荧光显微镜接收的图像包围整个动物(图2C)。在MIP图像中,应选择特定区域,如咽部(图3C)。
    6. 对于落射荧光图像,选择"分析"测量"以获得关于指定区域的任意单位(AU)的面积大小和平均像素强度(MPI)的总结。该信息将显示在单独的窗口中,并可以直接从数据分析软件中复制/粘贴,或者保存为Excel文件(* .xls)。
    7. 对于共焦图像,继续测量P体和SG数量和尺寸,必须确定分配单个像素或像素簇以定义荧光RNP颗粒的阈值。选择图像>调整>阈值和经验分配适当的最小/最大值(图3C)。该值将根据转基因,显微镜选择和成像设置而有所不同。一旦分配,这些值必须在每个分析中保持一致。
    8. 选择分析>分析粒子并勾选"显示结果","汇总"和"排除边缘",然后按OK(图3C)。
    9. 两个单独的窗口显示每个RNP颗粒的"结果",生成区域(颗粒大小)和平均值(MPI)以及包括"计数"(RNP颗粒数),"总面积"和"平均大小"的"摘要"。这些数据可以直接复制到数据分析软件和/或保存为excel文件。
  2. 统计分析
    1. 与每个菌株和条件检查的动物数量保持一致。
    2. 每个测定应重复至少三(3)次。
    3. 使用Mann Whitney或Wilcoxon Kruskal Wallis测试,其显着性截止水平 0.05用于不同组之间的比较,并使用Bonferroni或False Discovery Rate(FDR)进行多次成对比较。

笔记

已知P体和SGs在细胞中的大小和数量上基本上不同(Teixeira等人,2005)。测量变得更加精确,并且当专注于特定组织(例如,肌肉,咽或肠)中的定量和增加样品量时,最终的值代表较小的标准偏差。

食谱

  1. 磷酸盐缓冲液(1M)
    1. 对于1L,在蒸馏水中溶解102.2g KH 2 PO 4和57.06g K 2 HPO 4,填充1 L.这是一个1M溶液,pH 6.0
    2. 在121℃高压灭菌20分钟
    3. 在室温下存放
  2. 线虫生长培养基(NGM)琼脂平板
    1. 混合3g NaCl,2.5g细菌蛋白胨,0.2g链霉素,17g琼脂并加入900ml蒸馏水。在121℃高压灭菌20分钟
    2. 冷却至55-60°C
    3. 加入1ml胆固醇储备溶液,1ml 1M CaCl 2,1ml 1M MgSO 4,1ml制霉菌素储备溶液,25ml无菌1M磷酸盐缓冲液,pH 6.0,蒸馏无菌水至1升
    4. 移取每个培养皿8毫升培养基,并使其凝固
    5. 将板保存在4°C直到使用
  3. M9缓冲液
    1. 将3g KH 2 PO 4,6g Na 2 HPO 4,5L NaCl的1L蒸馏水溶解水
    2. 在121℃高压灭菌20分钟
    3. 冷却并加入1ml 1M MgSO 4(无菌)
    4. 在4°C存储M9缓冲液
  4. 5%琼脂糖垫
    1. 称重0.5 g琼脂糖,并加入圆柱形玻璃烧杯中
    2. 加入10ml M9缓冲液
    3. 在微波炉中加热混合物,直到接近沸腾。拿出来,用移液管吸头搅拌,再次煮沸。重复直到琼脂糖完全溶解
    4. 通过在中线放置一条磁带来修改两个显微镜幻灯片
    5. 在两个录像带之间放置一个空的显微镜载玻片(图4A)
    6. 将新鲜的5%琼脂糖溶液(约50μl)滴入载玻片的中部(图4B)
    7. 拿第四个显微镜载玻片放在琼脂糖滴上。轻轻按下压扁。胶带用作间隔物以使琼脂糖垫具有特定的厚度(图4C)
    8. 让琼脂糖硬化30秒,取出顶部显微镜载玻片
    9. 用手术刀将琼脂糖垫的边缘快速切成约20 x 20 mm的正方形
    10. 立即进行样品制备(见程序B),因为琼脂糖垫将在大约5分钟内开始干燥
      注意:将顶部显微镜载玻片作为盖子包含较长的湿度(约1小时)。因此,可以在实验过程中快速制备和使用几种琼脂糖垫

      图4.用于荧光成像的琼脂糖垫的制备。线虫。 A.两片玻璃片用带有侧面为空的显微镜载玻片的带子修改。 B.将一滴50μl5%琼脂糖溶液置于显微镜载玻片的中间(箭头)。 C.迅速将另一个显微镜载玻片放在上面,轻轻将其推下来,使琼脂糖下降。

致谢

这项工作是由欧洲研究委员会(ERC),欧洲委员会第七届支持框架计划的资助提供的。我们想要承认宾夕法尼亚州费城的方仁实验室,他们开发了用于长期成像的琼脂糖垫。线虫。

参考文献

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Rieckher, M. and Tavernarakis, N. (2017). P-body and Stress Granule Quantification in Caenorhabditis elegans. Bio-protocol 7(2): e2108. DOI: 10.21769/BioProtoc.2108.
  2. Rousakis, A., Vlanti, A., Borbolis, F., Roumelioti, F., Kapetanou, M. and Syntichaki, P. (2014). Diverse functions of mRNA metabolism factors in stress defense and aging of Caenorhabditis elegans. PLoS One 9(7): e103365.
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